Compounds with HIV-1 integrase inhibitory activity and use thereof as anti-HIV/AIDS therapeutics

ABSTRACT

Pharmacophore models to be used in drug design and discovery are provided. An in silico protocol and in vitro assays are presented. Compounds and their pharmaceutically acceptable salts with HIV-1 integrase inhibitory and anti-HIV activity and use thereof in the treatment of HIV/AIDS and related infections either alone or in combination with all the known antiretroviral therapeutics are described.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser. No. 60/911,446, filed on Apr. 12, 2007, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compounds with HIV-1 integrase inhibitory activity and their use in the treatment of HIV/AIDS and related infections.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus (type-1) (HIV-1) belongs to retrovirus and a causative virus of Acquired Immunodeficiency Syndrome (AIDS). The pol gene of HIV-1 encodes three essential enzymes: reverse transcriptase (RT), protease (PR) and integrase (IN). Currently 21 FDA approved drugs targeting RT and PR are available and are administered in various combinations. The combination of highly active antiretroviral therapy (HAART) in compliant patients comes close to stopping virus evolution, however, eradication of the infection has not been achieved because of the persistence of latent HIV-1 in resting memory CD4+ T cells. Moreover, several factors including the emergence of multidrug-resistant HIV strains, drug toxicity, the patient's ability to adhere to the prescribed therapy and expensive medication have necessitated a reason to develop novel drugs, which target other viral replication processes.

HIV-1 integrase has emerged as an attractive target for antiretroviral drugs because of its crucial role in the viral replication processes. HIV-1 integrase catalyses two crucial steps required for the integration of viral DNA into the host chromosome. In the first step, while in the cytoplasm of an infected cell, integrase selectively cleaves two nucleotides (GT) from the 3′ terminal of the viral cDNA in a reaction known as 3′-processing. Immediately after translocation to the nucleus as a component of the pre-integration complex, integrase randomly inserts the viral cDNA into the host genome, and this reaction is referred to as strand transfer or integration. Over the past years several integrase inhibitors have been discovered, yet none of them reached the clinic.

SUMMARY OF THE INVENTION

In view of the foregoing, the addition of integrase inhibitors to the existing combination therapy would certainly improve the outcome of the HIV/AIDS treatment.

Pharmacophore Models in Drug Design and Discovery:

Once a potential inhibitor against a known target has been identified, computational approaches such as pharmacophore-based three-dimensional (3D) database searching can play a key role in the discovery of novel leads with different chemical scaffolds.

A pharmacophore refers to the three dimensional arrangement of various functional groups (chemical features) that is essential for the molecule to bind effectively against a specific enzyme, protein, or a receptor.

Accordingly, the invention features the following:

1. Pharmacophore models that are shown in FIGS. 4-7 and pharmacophore features combination given in Tables 1-4.

2. Compounds represented by Formula 1-40 and their pharmaceutically acceptable salts with HIV-1 integrase inhibitory and anti-HIV activity and uses thereof in the treatment of HIV/AIDS and related infections alone and in combination with all the known antiretroviral therapeutics.

Representative compounds exemplified by Formula 1 with their integrase inhibitory activities are given in Table 5. R₁: A variety of substitutions including (not limited to) hydrogen, halogens, hydroxyl, sulfhydryl, alkoxy, carboxyl, nitro, cyano, amino, amido, sulfonyl or any other organic functional group containing any number of carbon atoms. R₂: Aliphatic or aromatic groups including alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl. Representative substitutions include (not limited to) halo, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl and substituted heterocyclics. n=C1-C4

Representative compounds exemplified by Formula 2 with their integrase inhibitory activities are given in Table 6. R₁: Aliphatic, aromatic, heterocyclic groups including alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl. Representative substitutions include (not limited to) halo, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted heterocyclics. R₂: A variety of cyclic (3-7 member) aliphatic, aromatic, heterocyclic groups optionally substituted by functional groups including (not limited to) halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide. X═O or S or N

Representative compounds exemplified by Formula 3 with their integrase inhibitory activities are given in Table 7. R₁: A variety of cyclic (1-7 member) or acyclic (1-7 member) aliphatic, aromatic, heterocyclic groups optionally substituted by functional groups including (not limited to) halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide.

Representative compounds exemplified by Formula 4 with their integrase inhibitory activities are given in Table 8. R₁, R₁′: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy (Symmetric or asymmetric substitutions). R₂, R₂′: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy, and a variety of alkyl, aryl, heteroaryl groups optionally substituted with functional groups including (not limited to) halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide.

Representative compounds exemplified by Formula 5 with their integrase inhibitory activities are given in Table 9. R₁: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy, NH-with optionally substituted aliphatic, heteroaliphatic, aryl, heteroaryl groups. X—R₂: C—R₂′ N—R₂ R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₃-R₆: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. Z═O, S

Representative compounds exemplified by Formula 6 are given in Table 10. R₁: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₃: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy. X═C or N; γ=C or N; Z C or N

Representative compounds exemplified by Formula 7 are given in Table 11. R₁: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₂: Hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, aryloxy. R₃: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy. X═C or N; γ=C or N; Z═C or N

Representative compounds exemplified by Formula 8 are given in Table 12. R₁: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₂: Hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, aryloxy R₃: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy. X═C or N; γ=C or N; Z═C or N

Representative compounds exemplified by Formula 9 are given in Table 13. R₁: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₃: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy, X═C or N; Y═C or N; Z═C or N

Representative compounds exemplified by Formula 10 are given in Table 14 R₁-R₅: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₅: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy

Representative compounds exemplified by Formula 11 are given in Table 15 R₁: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy R₂-R₄: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 12 are given in Table 16 R₁ and R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₂: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy

Representative compounds exemplified by Formula 13 are given in Table 17 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 14 are given in Table 18 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. X═N or O or S

Representative compounds exemplified by Formula 15 are given in Table 19 R₂: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy R₁ and R₃-R₄: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 16 are given in Table 20 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 17 are given in Table 21 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 18 are given in Table 22 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 19 are given in Table 23 R₁ and R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₂: Hydrogen, hydroxyl, carboxyl, carbonyl, sulfhydryl, alkoxy, aryloxy

Representative compounds exemplified by Formula 20 are given in Table 24 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 21 are given in Table 25 R₁ and R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 22 are given in Table 26 R₁: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₂: Hydrogen, hydroxyl, carboxyl, carbonyl, sulfhydryl, alkoxy, aryloxy

Representative compounds exemplified by Formula 23 are given in Table 27 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 24 are given in Table 28 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 25 are given in Table 29 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 26 are given in Table 30 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 27 are given in Table 31 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 28 are given in Table 32 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 29 are given in Table 33 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 30 are given in Table 34 R₂: Hydrogen, hydroxyl, carboxyl, carbonyl, sulfhydryl, alkoxy, aryloxy R₁ and R₂-R₄: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. X═N or O or S

Representative compounds exemplified by Formula 31 are given in Table 35 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 32 are given in Table 36 R₁ and R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. R₂: Hydrogen, hydroxyl, carboxyl, carbonyl, sulfhydryl, alkoxy, aryloxy

Representative compounds exemplified by Formula 33 are given in Table 37 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 34 are given in Table 38 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 35 are given in Table 39 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. X═N or O or S

Representative compounds exemplified by Formula 36 are given in Table 40 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 37 are given in Table 41 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 38 are given in Table 42 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 39 are given in Table 43 R₁-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics.

Representative compounds exemplified by Formula 40 are given in Table 44 R₁-R₅: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. X═O or S or N

Representative compounds exemplified by Formula 41 are given in Table 45 R₁-R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatics, heterocyclics. X═C or N; Y═C or N; Z═C or N 3. Pharmacophore features combination are given in Table 46. 4. Compounds represented by Formula 42-49 and their pharmaceutically acceptable salts with HIV-1 integrase inhibitory and anti-HIV activity and use thereof in the treatment of HIV/A/DS and related infections alone and incombination of all the known antiretroviral therapeutics.

Representative compounds exemplified by Formula 42 are given in Tables 47 and 48 R₁: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Aliphatic amides with optional substitutions. Hydrazides or hydrazines with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatic, heterocyclic groups. R₂: Hydrogen, hydroxyl, sulfhydryl, alkoxy, aryloxy R₅: Hydrogen, halogen, hydroxyl, carboxyl, alkoxy, aryloxy, amine, amide, nitro, sulfonamide, substituted aromatics or heterocyclic groups. X═O or S or NH

Representative compounds exemplified by Formula 43 are given in Table 49 R₁: Hydrogen atom, substituted cyclic or acyclic aliphatic or heteroaliphatic groups, substituted aromatic or heteroaromatic 4-7 member rings of carbon, nitrogen, oxygen, and sulfur atoms. Substitutions include (not limited to) hydrogen, halogen, alkoxy, aryloxy, sulfhydryl, sulfonamide, sulfone, carboxyl, amide, amine and nitro groups. R₂: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatic, heterocyclic groups. R₃: Hydrogen atom, hydroxyl, alkoxy, aryloxy, halogen, amine, carboxyl, amide, and nitro.

R₁: Hydrogen atom, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amide, sulfonyl, sulfonamide and nitro. Substituted aliphatic, heteroaliphatic, aryl, and heteroaryl groups. X═C or N

R₁: Hydrogen atom, optionally substituted aliphatic, heteroaliphatic, aryl and heteroaryl groups. Optional substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amide, sulfonyl, sulfonamide and nitro. R₂: Hydrogen atom, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amide, sulfonyl, sulfonamide and nitro. Substituted aliphatic, heteroaliphatic, aryl and heteroaryl groups.

More specifically, the invention provides a composition comprising a compound, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the compound conforms to one of the pharmacophores shown in FIGS. 4-7 and Tables 1-4 and 46. The compound may be of any of Formulas 1-49. For example, the compound is selected from the group consisting of the compounds shown in Tables 5-45 and 47-57. The composition may further comprise a pharmaceutically acceptable carrier.

Representative compounds exemplified by Formula 44 are given in Table 50 R₁: Hydrogen atom, hydroxyl, sulfhydryl, carboxyl, amine, alkoxy, arloxy, and amide. R₂-R₃: Hydrogen atom, cyclic or acyclic, straight or branched, saturated or unsaturated aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatic, heterocyclic groups X═O or S

Representative compounds exemplified by Formula 45 are given in Table 51 R₁-R₂: Hydrogen atom, aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatic, heterocyclic groups X═C or N Y═C or N

Representative compounds exemplified by Formula 46 are given in Table 52. R₁: Carboxyl, or bioisosteres of carboxyl group such as triazole, tetrazole groups R₂-R₃: Hydrogen atom, aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatic, heterocyclic groups. X═O or S Several structurally diverse compounds retrieved by pharmacophore model 1 (HT5NODF.01) with HIV-1 integrase inhibitory activity are give in Table 53.

Representative compounds exemplified by Formula 47 are given in Table 54 R₁: Carboxyl, or bioisosteres of carboxyl group such as triazole, oxazole, and tetrazole groups. R₂: Hydrogen atom, aliphatic, heteroaliphatic groups with optional substitutions, aryl or heteroaryl groups with optional substitutions. Representative substitutions include (not limited to) hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amido, sulfonyl, sulfonamide and substituted aromatic, heterocyclic groups.

The invention also provides a method of inhibiting HIV-1 integrase, comprising contacting a composition of claim 1 with an HIV-1 integrase, thereby inhibiting the activity of the HIV-1 integrase.

The invention further provides a computer-readable medium comprising a representation of one of the pharmacophores shown in FIGS. 4-7 and Tables 1-4 and 46.

Moreover, the invention provides a method of identifying an HIV integrase inhibitor, comprising comparing the three-dimensional structure of a compound with the three-dimensional structure of one of the pharmacophores shown in FIGS. 4-7 and Tables 1-4 and 46, and selecting the compound if the compound conforms to the features of the pharmacophore.

The above-mentioned and other features of this invention and the manner of obtaining and using them will become more apparent, and will be best understood, by reference to the following description, taken in conjunction with the accompanying drawings. The drawings depict only typical embodiments of the invention and do not therefore limit its scope.

DESCRIPTION OF THE FIGURES

FIG. 1. In silico screening protocol implemented in the discovery of novel inhibitors for HIV-1 integrase.

FIG. 2. Structures of the β-diketoacid bioisoteres used in the generation of common feature pharmacophore hypotheses.

FIG. 3. Structures of the β-diketoacid inhibitors of HIV-1 Integrase used in the generation of common feature pharmacophore hypotheses.

FIG. 4. (A) The best ranked four featured pharmacophore hypothesis (ECPHM4.01). ECPHM4.01 was used in the database mining to identify novel inhibitors for REV-1 integrase. (B) S-1360 is mapped on to ECPHM4.01. Pharmacopore features are color coded; Hydrophobic Aromatic (HYA)-light blue, H-Bond Acceptor (HBA)-green, H-Bond Donor (HBD)-magenta.

FIG. 5. (A) The best ranked four featured pharmacophore hypothesis (HCT4a.01). Pharmacophore hypotheses (HCT4a.01-10) were generated using conformations of the training set compounds (1-4) similar to the crystallographically determined structure of one of the training set compounds. HCT4a.01 was also used in the database mining to identify novel inhibitors for HIV-1 integrase. (B) S-1360 is mapped on to HCT4a.01. Pharmacopore features are color coded; Hydrophobic Aromatic (HYA)-light blue, H-Bond Acceptor (HBA)-green, H-Bond Donor (HBD)-magenta.

FIG. 6. (A) The best ranked four featured pharmacophore hypothesis (HSCT6AF25.01). All the chemical features found in the training set compounds were used in the pharmacophore hypotheses generation. (B) One of the training set compounds is mapped on to HSCT6AF25.01. Pharmacopore features are color coded; Hydrophobic Aromatic (HYA)-light blue, H-Bond Acceptor (HBA)-green, H-Bond Donor (HBD)-magenta, Negatively Ionizable (NI)— dark blue.

FIG. 7. (A) The best ranked four featured pharmacophore hypothesis (HSCT6NoN25.01). All the chemical features found in the training set compounds except negatively ionizable feature were used in the pharmacophore hypotheses generation. (B) One of the training set compounds is mapped on to HSCT6NoN25.01. Pharmacopore features are color coded; Hydrophobic Aromatic (HYA) light blue, H-Bond Acceptor (HBA)-green, H-Bond Donor (HBD)-magenta.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based, at least in part, upon the unexpected discovery that the compounds identified as described below inhibit the activity of HIV-1 integrase. Accordingly, the invention provides a composition comprising a compound, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the compound conforms to one of the pharmacophores shown in FIGS. 4-7 and Tables 1-4 and 46. The compound may be of any of Formulas 1-49. For example, the compound is selected from the group consisting of the compounds shown in Tables 5-45 and 47-57.

A composition of the invention may further comprise a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carriers” include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

A compound of the invention may be obtained by chemical synthesis or from commercial sources. The composition of the invention is useful for inhibiting HIV-1 integrase and treating HIV-1/AIDS.

A composition of the invention is formulated to be compatible with its intended route of administration. See, e.g., U.S. Pat. No. 6,756,196. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, sterile water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the compounds in the required amounts in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the compounds into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the compounds can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically or cosmeceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compositions are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compositions of the invention can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the compositions are prepared with carriers that will protect the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically or cosmeceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form,” as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic or cosmeceutic effect in association with the required pharmaceutical or cosmeceutical carrier.

One object of the invention is to provide a method for inhibiting HIV-1 integrase in vitro or in vivo. The method comprises contacting a composition of the invention with an HIV-1 integrase.

Accordingly, in one embodiment, a composition of the invention is contacted with an HIV-1 integrase, e.g., in a cell, thereby inhibiting the activity of the HIV-1 integrase.

The invention further provides for both prophylactic and therapeutic methods of treating a subject in need thereof (e.g., a subject with HIV-1/AIDS) an effective amount of a composition of the invention.

“Subject,” as used herein, refers to a human or animal, including all vertebrates, e.g., mammals, such as primates (particularly higher primates), sheep, dog, rodents (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, cow; and non-mammals, such as chicken, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an animal.

A subject to be treated may be identified, e.g., using diagnostic methods known in the art, as being suffering from or at risk for developing a disease or condition. The subject may be identified in the judgment of a subject or a health care professional, and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.

An “effective amount” is an amount of a therapeutic agent that is capable of producing a medically desirable result as delineated herein in a treated subject. The medically desirable result may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).

Toxicity and therapeutic efficacy of a compound of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of a compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

A therapeutically effective amount of the compounds (I.e., an effective dosage) may range from, e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. The compounds can be administered, e.g., one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. In subjects suffering from chronic diseases, such as arthritis or osteoporosis, life-long treatment may be necessary, for example, one time every day or preferably one time per week. It is furthermore understood that appropriate doses of a compound depend upon the potency of the compound. When one or more of these compounds is to be administered to a subject (e.g., an animal or a human), a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, the severity of the disease or disorder, previous treatments, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compounds can include a single treatment or, preferably, can include a series of treatments.

Another object of the invention is to provide a method of identifying HIV-1 integrase inhibitors. Accordingly, the invention provides a computer-readable medium comprising a representation of one of the pharmacophores shown in FIGS. 4-7 and Tables 1-4 and 46.

As used herein, “computer readable media” refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to, magnetic storage media, such as floppy discs, hard disc storage media, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily create a computer readable medium having recorded thereon a representation of a pharmacophore of the invention using any of the methods well known in the art.

By providing a representation of a pharmacophore of the invention in computer readable form, a skilled artisan can routinely access the pharmacophore information for a variety of purposes. For example, one skilled in the art can use a pharmacophore of the invention in computer readable form to compare with compound information stored within data storage means. Search means are used to identify compounds that match the features of the pharmacophore and therefore are candidate HIV-1 integrase inhibitors.

Accordingly, the invention provides a method of identifying an HIV-1 integrase inhibitor. The method comprises comparing the three-dimensional structure of a compound with the three-dimensional structure of a pharmacophore of the invention, and selecting the compound if the compound conforms to the features of the pharmacophore.

The following examples are intended to illustrate, but not to limit, the scope of the invention. While such examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.

EXAMPLES

The in silico protocol followed in the discovery of compounds with HIV-1 integrase inhibitory activity is schematically shown in FIG. 1.

Pharmacophore Models

Several common feature pharmacophore models were generated using two sets of known HIV-1 integrase inhibitors (FIGS. 2-3). The top-ranking pharmacophores are expected to identify the hypothetical orientation of the active compounds and the common binding features interacting with the target. All training set inhibitors were somewhat structurally diverse but possessed some . . . common chemical features, and comparable inhibitory potencies. The pharmacophore models were generated using low energy conformation as well as using conformations similar to the crystallographically determined conformation of known integrase inhibitors. On the basis of the structural information from these known inhibitors and the active site of IN, a set of features were selected to be present in the pharmacophore generation experiment. The chemical features considered in the pharmacophore model generation run were H-bond donor (HBD), H-bond acceptor (HBA), Hydrophobic aromatic (HYA), Aromatic ring center (AR), and Negatively ionizable (NT) features. The validated pharmacophore models that were used in the database search are shown in FIGS. 4-7. The most potent integrase inhibitors were mapped onto the best ranked pharmacophore models (FIGS. 4-7). All the pharmacophore models that were generated specific to HIV-1 integrase are given in Tables 1-4 along with feature combinations and ranking scores.

Database Search:

The highest ranked common feature pharmacophore models (FIGS. 4-7) were used as search queries to retrieve compounds with novel chemical structure and desired chemical features from an in-house multi-conformer Catalyst-formatted database consisting of ˜5,000,000 compounds. Drug-like properties of the retrieved hits from the database search were calculated using Accord for Excel.

Docking and Virtual Screening:

The subunit B of the core domain X-ray structure of Integrase (PDB 1BIS) in which all the active site amino acid residues were resolved was chosen for our docking and virtual screening purpose. A Mg²⁺ ion was placed in the active site between carboxylate oxygen atoms of amino acid residues D64 and D116 considering the geometry of the Mg²⁺ ion that was present in the subunit A of IN in PDB 1BIS and subunit A in IN-5CITEP complex X-ray structure (PDB 1SQ4). All the water molecules present in protein were removed and proper protonation states were assigned for acidic and basic residues of the protein. Docking was preformed using version 1.2 of the GOLD (Genetic Optimization for Ligand Docking) software package. GOLD is an automated ligand docking program that uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the receptor. The algorithm was tested on a dataset of over 300 complexes extracted from the Brookhaven Protein DataBank. GOLD succeeded in more than 70% cases in reproducing the experimental bound conformation of the ligand. GOLD requires a user defined binding site. It searches for a cavity within the defined area and considers all the solvent accessible atoms in the defined area as active site atoms. A 20 Å radius active site was defined considering the carboxylate oxygen (OD1) atom of residue D64 as the center of the active site. All the compounds retrieved by the pharmacophore models were docked into the active site of IN. On the basis of the GOLD fitness score, for each molecule a bound conformation with high fitness score was considered as the best bound-conformation. All docking runs were carried out using standard default settings with a population size of 100, a maximum number of 100,000 operations, and a mutation and crossover rate of 95. The fitness function that was implemented in GOLD consisted basically of H-bonding, complex energy and ligand internal energy terms.

Materials. Chemicals, and Enzymes:

All compounds were dissolved in DMSO and the stock solutions were stored at −20° C. The γ[³²P]-ATP was purchased from either Amersham Biosciences or ICN.

Preparation of Oligonucleotide Substrates

The oligonucleotides 21 top, 5′-GTGTGGAAAATCTCTAGCAGT-3′ (SEQ ID NO:1) and 21 bot, 5′-ACTGCTAGAGATTTTCCACAC-3′ (SEQ ID NO:2) were purchased from Norris Cancer Center Core Facility (University of Southern California) and purified by UV shadowing on polyacrylamide gel. To analyze the extent of 3′-processing and strand transfer using 5′-end labeled substrates, 21 top was 5′-end labeled using T4 polynucleotide kinase (Epicentre, Madison, Wis.) and γ [³²P]-ATP (Amersham Biosciences or ICN). The kinase was heat-inactivated and 21 bot was added in 1.5-molar excess. The mixture was heated at 95° C., allowed to cool slowly to room temperature, and run through a spin 25 mini-column (USA Scientific) to separate annealed double-stranded oligonucleotide from unincorporated material.

Integrase Assays:

To determine the extent of 3′-processing and strand transfer, wild-type IN was preincubated at a final concentration of 200 nM with the inhibitor in reaction buffer (50 mM NaCl, 1 mM HEPES, pH 7.5, 50 μM EDTA, 50 μM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/ml bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide, and 25 mM MOPS, pH 7.2) at 30° C. for 30 min. Then, 20 nM of the 5′-end ³²P-labeled linear oligonucleotide substrate was added, and incubation was continued for an additional one hour. Reactions were quenched by the addition of an equal volume (16 μl) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue). An aliquot (5 μl) was electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M tris-borate pH 8.3, 2 mM EDTA, 20% acrylamide, 8M urea).

Gels were dried, exposed in a PhosphorImager cassette, and analyzed using a Typhoon 8610 Variable Mode Imager (Amersham Biosciences) and quantitated using ImageQuant 5.2. Percent inhibition (% I) was calculated using the following equation: %I=100×[1−(D−C)/(N−C)]

where C, N, and D are the fractions of 21-mer substrate converted to 19-mer (3′-processing product) or strand transfer products for DNA alone, DNA plus IN, and IN plus drug, respectively. The IC₅₀ values were determined by plotting the logarithm of drug concentration versus percent inhibition to obtain concentration that produced 50% inhibition.

Anti-HIV Assays in Cultured Cells:

The anti-HIV activity was evaluated in human T cell line CEM-SS infected with HIV-1 as described by Weislow et al. 45 In brief, cells were plated in 96-well plates at 5×10³ cells/well and infected with HW-1RF (MOI=0.3). Serial dilutions of compounds were then immediately added to the cells in a final volume of 200 μl. In each experiment, AZT and dextran sulfate were included as control compounds for anti-HIV activity. The cells were maintained at 37° C. with 5% C02-containing humidified air for 6 days. Cell viability was quantified by absorbance at 450 nm after 4 h incubation with 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) at 0.2. mg/ml. Antiviral activity was graded based on the degree of anti-HIV protection as active (80-100% protection), moderate (50-79% protection) and inactive (0-49% protection). Toxicity of the compounds was determined simultaneously on the same plate in uninfected CEM-SS cells.

TABLE 1 Pharmacophore models (ECPHM4) generated using the training set compounds (1-4) shown in FIG. 1 (Conformations used are low energy conformations generated using Catalyst (Accelrys, Inc) program) Hypothesis Features Combinations ^(a) Ranking Score ECPHM4.01 HYA, HBD1, HBD2, HBA 35.02 ECPHM4.02 HYA, HBD1, HBD2, HBA 35.02 ECPHM4.03 HYA, HBD1, HBA, HBA1 34.22 ECPHM4.04 HYA, HBD1, HBA, HBA1 34.22 ECPHM4.05 HYA, HBD1, HBD2, HBA 34.08 ECPHM4.06 HYA, HBD1, HBD2, HBA 34.08 ECPHM4.07 HYA, HBD1, HBD2, HBA 33.76 ECPHM4.08 HYA, HBD1, HBA, HBA1 33.41 ECPHM4.09 HYA, HBD1, HBA, HBA1 33.41 ECPHM4.10 HYA, HBD1, HBA, HBA1 33.41 ^(a) HYA—Hydrophobic Aromatic; HBA—H-Bond Acceptor; HBD—H-Bond Donor Feature

TABLE 2 Pharmacophore models (HCT4a) generated using the training set compounds (1-4) shown in FIG. 1 (conformations used are similar to crystallographically determined conformation of one of the training set compounds) Hypothesis Features Combinations ^(a) Ranking Score HCT4a.01 HYA, HBD, HBA1, HBA2 37.41 HCT4a.02 HYA, HBD, HBA1, HBA2 37.20 HCT4a.03 HYA ,HBD, HBA1, HBA2 37.20 HCT4a.04 HYA, HBD, HBA1, HBA2 37.20 HCT4a.05 HYA, HBD, HBA1, HBA2 36.52 HCT4a.06 HYA, HBD, HBA1, HBA2 36.46 HCT4a.07 HYA, HBD, HBA1, HBA2 36.28 HCT4a.08 HYA, HBD, HBA1, HBA2 35.45 HCT4a.09 HYA, HBD, HBA1, HBA2 35.45 HCT4a.10 HYA, HBD, HBA1, HBA2 35.24 ^(a) HYA—Hydrophobic Aromatic; HBA—H-Bond Acceptor; HBD—H-Bond Donor Feature

TABLE 3 Pharmacophore models generated using the training set compounds (5-10) shown in FIG. 2 (All chemical features found in the training set compounds considered for pharmacophore generation) Hypothesis Features Combinations ^(a) Ranking Score HSCT6AF25.01 HYA, HYA1, HBD, HBA, NI 88.09 HSCT6AF25.02 HRA, HYA, HBD, HBA, NI 87.68 HSCT6AF25.03 HRA, HYA, HBD, HBA, NI 87.68 HSCT6AF25.04 HRA, HYA, HBD, HBA, NI 87.09 HSCT6AF25.05 HRA, HYA, HBD, HBA, NI 87.09 HSCT6AF25.06 HYA, HYA1, HBA, HBA1, NI 86.89 HSCT6AF25.07 HRA, HRA1, HBD, HBA, NI 86.65 HSCT6AF25.08 HRA, HRA1, HBD, HBA, NI 86.65 HSCT6AF25.09 HYA, HYA1, HBA, HBA1, NI 86.48 HSCT6AF25.10 HYA, HYA1, HBA, HBA1, NI 86.48 ^(a) HRA—Ring Aromatic; HYA—Hydrophobic Aromatic; HBA—H-Bond Acceptor; HBD—H-Bond Donor; NI—Negatively Ionizable Feature

TABLE 4 Pharmacophore models generated using the training set compounds (5-10) shown in FIG. 2 (All chemical features found in the training set compounds considered for pharmacophore generation except the negatively ionizable (NI) feature) Hypothesis Features Combinations ^(a) Ranking Score HSCT6NoNF25.01 HYA, HYA1, HBD, HBA, HBA1 80.97 SCT6NoNF25.02 HRA, HYA, HBD, HBA, HBA1 80.52 SCT6NoNF25.03 HRA, HYA, HBD, HBA, HBA1 80.52 SCT6NoNF25.04 HYA, HYA1, HBD, HBA, HBA1 80.28 SCT6NoNF25.05 HRA, HYA, HBD, HBA, HBA1 80.22 SCT6NoNF25.06 HRA, HYA, HBD, HBA, HBA1 80.22 SCT6NoNF25.07 HRA, HYA, HBD, HBA, HBA1 79.97 SCT6NoNF25.08 HRA, HYA, HBD, HBA, HBA1 79.94 SCT6NoNF25.09 HYA, HYA1, HBD, HBA, HBA1 79.83 SCT6NoNF25.10 HYA, HYA1, HBA, HBA1, HBA2 79.77 ^(a) HRA—Ring Aromatic; HYA—Hydrophobic Aromatic; HBA—H-Bond Acceptor; HBD—H-Bond Donor Feature

TABLE 5 Inhibition of HIV-1 Integrase Catalytic Activities of Representative Compounds Exemplified by Formula 1

Inhibition of IN Catalytic Activities IC₅₀ (μM) 3′- Strand GOLD Example R₁ R₂ n processing Transfer Score  X92 3-COOH, 4-OH

2 17 ± 13 11 ± 6 52.18  X94 3-COOH, 4-OH

2 15 ± 10 17 ± 11 57.90 X169 3-COOH, 4-OH

3 44 ± 20 35 ± 16 52.59 X170 3-COOH, 4-OH

2 15 ± 3 11 ± 5 51.57 X172 3-COOH, 4-OH

2 38 ± 16 38 ± 16 56.39 X185 3-COOH, 4-OH

2 36 ± 23 23 ± 3 57.25 X175 3-OH, 4-COOH

3 32 ± 14 25 ± 5 54.57 X188 3-OH, 4-COOH

2 33 ± 23 23 ± 13 57.62 X192 3-OH, 4-COOH

2 61 ± 34 17 ± 4 57.46  X95 3-COOH

2 93 ± 12 >100 51.98 X100 3-COOH

2  100 >100 56.15  X26 3-COOH

2 >100 >100 53.43 X173 4-COOH

2 97 ± 6 85 ± 15 50.05 X176 4-COOH

2 86 ± 15 86 ± 17 51.26  X56 3-COOH

2 >100 >100 55.78  X91 3-COOH

1 >100 >100 55.06  X89 3-COOH

1 >100 >100 52.07  X93 3-COOH

1 >100 >100 58.95  X97 3-COOH

1 98 ± 4 >100 56.04 X118 4-OH

2 99 ± 3 100 43.93 X177 3-OH

2 >100 >100 47.81 X190 3-OH

2 >100 90 ± 10 53.28 X184 2-OH, 4-NO₂

1 >100 >100 49.84 X191 2-OH, 5-NO₂

1 84 ± 24 80 ± 18 47.49 X193 2-OH, 4-NO₂

1 >100 >100 46.44 X194 2-OH, 5-NO₂

1 20 ± 7 18 ± 11 49.77 X171 2-OH, 5-Cl

1 32 ± 11 17 ± 2 46.78 X195 2-OH, 4-NO₂

1 >100 >100 X102

59 ± 31 75 58.64 X103

59 ± 37 69 51.30

TABLE 6 Inhibition of HIV-1 Integrase Catalytic Activities of Representative Compounds Exemplified by Formula 2 Inhibition of IN Catalytic Activities IC₅₀ (μM) Strand GOLD Example Structure 3'-processing Transfer Score X200

>100 >100 46.03 X124

83 ± 21 44 ± 16 50.69 X174

>100 >100 50.89 X178

98 ± 5    100 54.21 X187

97 ± 6  93± 12 56.55 X105

88 ± 22 84 ± 2  59.21 X90

>100 >100 46.41 X220

52 ± l1 27 ± 6  47.27 X221

>100 >100 36.51 X223

70 ± 27 38 ± 14 46.41 X281

>100   100 50.30 OT35

41 ± 11 38 ± 16 51 OT3

>100 92 ± 7  50 OT4

48 ± 7  22 ± 4  53 OT5

>>100   54 ± 17 47 OT6

17 ± 4  13 ± 7  52 OT9

>100 97 ± 5  51 OT17

42 ± 20 27 ± 11 51 OT35

41 ± 11 38 ± 16 51 OT37

>100 79 ± 8  50 RT23

>100 21

TABLE 7 Inhibition of HIV-1 Integrase Catalytic Activities of Representative Compounds Exemplified by Formula 3 Inhibition of IN Catalytic Activities IC₅₀ (μM) Strand GOLD Example Structure 3'-processing Transfer Score X71

>100 >100 44.21 X63

  100   100 48.80 X108

>100 >100 43.25 X114

40 ± 10 43 ± 6 54.73 X117

61 ± 10 32 ± 1 50.37 X121

13 ± 6  12 ± 8 54.07 X125

66 ± 6  44 ± 4 41.98 X132

16 ± 5  8 ± 1 52.06 X181

96 ± 7   73 ± 24 36.46 X196

78 ± 39  75 ± 43 47.78 X199

>100 >100 49.36

TABLE 8 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 4 HIV-1 integrase Inhibitory Activities IC₅₀ (μM) GOLD Example Structure 3'-processing Strand transfer Score X7

9 7 53 X8

28 21 51 X19

92 100 X35

100 94

TABLE 9 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 5 HIV-1 integrase Inhibitory Activities IC₅₀ (μM) GOLD Example Structure 3'-processing Strand transfer Score X228

40 16 50 XR8

50 39 XR7

83 45 XR14

15 17 XR13

11 13 XR10

72 32 XR6

9 9 XR15

100 9.5 XR3

100 25 XR5

74 19 XR12

81 28 LM11

56 15 XR44

28 26 LM33

100 36 NXR

TABLE 10 Representative Compounds Exemplified by Formula 6 GOLD Example Structure Score NXR1

14 NXR2

24 NXR3

23 NXR4

35 NXR5

23 NXR6

39 NXR7

23 NXR8

40 NXR9

37 NXR10

21 NXR11

21 NXR12

37 NXR13

34 NXR14

36

TABLE 11 Representative Compounds Exemplified by Formula 7 GOLD Example Structure Score COXR1

33 COXR2

42 COXR3

43 COXR4

49 COXR5

43 COXR6

41 COXR7

37 COXR8

45 COXR9

44 COXR10

46 COXR11

42 COXR12

47 COXR13

49 COXR14

52 COXR15

41 COXR16

45

TABLE 12 Representative Compounds Exemplified by Formula 8 GOLD Example Structure Score CHOXR1

35 CHOXR2

38 CHOXR3

41 CHOXR4

43 CHOXR5

44 CHOXR6

44 CHOXR7

41 CHOXR8

39 CHOXR9

40 CHOXR10

43 CHOXR11

42 CHOXR12

44 CHOXR13

42 CHOXR14

39 CHOXR15

42

TABLE 13 Representative Compounds Exemplified by Formula 9 GOLD Example Structure Score MXR1

33 MXR2

26 MXR3

31 MXR4

29 MXR5

36 MXR6

30 MXR7

33 MXR8

32 MXR9

35 MXR10

29 MXR11

41 MXR12

38 MXR13

53 MXR14

48 MXR15

48 MXR16

43 MXR17

48 MXR18

42 MXR19

42 MXR20

42 MXR21

49 MXR22

49 MXR23

35 MXR24

32 MXR25

32 MXR26

30

TABLE 14 HIV-1 IN Inhibitory Activities of Representative Compounds Exemplified by Formula 10 HIV-1 IN Inhibitory Activities IC₅₀ (μM) Strand GOLD Example Structure 3'-processing transfer Score ROM3

>12 12 28 ROM3A1

>100 >100 32 ROM3A2

94 98 28 ROM3A3

>100 >100 33 ROM3A4

64 59 42 ROM3A5

>100 >100 38 ROM3A7

>100 95 27 ROM3A6

>100 >100 32 ROM3A8

78 74 36 ROM3A9

80 53 43 ROM3A10

93 92 40 ROM3A11

91 90 59 SR9

>100 >100 39

TABLE 15 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 11 HIV-1 IN Inhibitory Activities Example Structure 3'-processing Strand transfer ROM1

78 20 ANA1CD

ANA2CD

ANA3CD

ANA4CD

ANA5CD

ANA6-AS

TABLE 16 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 12 HIV-1 IN Inhibitory Activities IC₅₀ (μM) GOLD Example Structure 3'-processing Strand transfer Score AV15

9 4 34 AV30

10 4 X114

40 43 45 SA1

59 9 45 NST20

100 68 55 NAV1

48 NAV2

56 NAV3

45 NAV4

40 NAV5

42 NAV6

41 NAV7

50 NAV8

50 NAV9

45 NAV10

42 NAV11

48 NAV12

48

TABLE 17 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 13 HIV-1 IN Inhibitory Activities GOLD Example Structure 3'-processing Strand transfer Score X155

93 67 51 X81

84 69 X36

>100 >100 T38

25 20 MMC8

>1000 395

TABLE 18 Inhibition of HIV-1 Integrase Catalytic Activities of Representative Compounds Exemplified by Formula 14 Inhibition of IN catalytic activities, IC₅₀ (μM) Example Structure 3'-processing Strand transfer RT35

90 19 RT42

>100 >100

TABLE 19 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 15 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X88

>100 79 61

TABLE 20 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 16 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X111

88 68 55

TABLE 21 HIV-1 Integrase Inhibitoiy Activities of Representative Compounds Exemplified by Formula 17 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X115

42 28 50

TABLE 22 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 18 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X134

>100 90 51

TABLE 23 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 19 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X143

30 10 53 X144

78 29

TABLE 24 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 20 HIV-1IN Inhibitory Activities Example Structure 3′-processing Strand transfer X148

89 90

TABLE 25 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 21 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X159

90 91 47

TABLE 26 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 22 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X160

16 17 46.08

TABLE 27 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 23 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X182

88 56 52

TABLE 28 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 24 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X205

57 28 52

TABLE 29 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 25 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X208

97 61 43

TABLE 30 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 26 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X218

76 56 40

TABLE 31 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 27 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X219

89 31 52

TABLE 32 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 28 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X234

90 23 55 X231

60 38 42

TABLE 33 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 29 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X244

88 68 55

TABLE 34 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 30 HIV-1IN Inhibitory Activities Example Structure 3′-processing Strand transfer X255

61 10 XR16

<100 <100 XR17

6 7 XR18

<100 <100 XR19

<100 <100 XR21

14 6 XR22

<100 <100 X257

99 80

TABLE 35 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 31 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X272

34 24 59

TABLE 36 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 32 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X278

89 38 49

TABLE 37 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 33 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X279

95 59 61

TABLE 38 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 34 HIV-1IN Inhibitory Activities GOLD Example Structure 3′-processing Strand transfer Score X280

>1000 85 54

TABLE 39 Inhibition of HIV-1 Integrase Catalytic Activities and GOLD Score of Representative Compounds Exemplified by Formula 35 Inhibition of IN catalytic activities, IC₅₀ (μM) GOLD Example Structure 3′-processing Strand transfer Score OT51

36 ± 29 14 ± 4 56

TABLE 40 Inhibition of HIV-1 Integrase Catalytic Activities and GOLD Score of Representative Compounds Exemplified by Formula 36 Inhibition of IN catalytic activities, IC₅₀ (μM) GOLD Example Structure 3′-processing Strand transfer Score OT27

69 ± 15 47 ± 22 38 OT24

98 ± 5  71 ± 23 36

TABLE 41 Inhibition of HIV-1 Integrase Catalytic Activities and GOLD Score of Representative Compounds Exemplified by Formula 37 Inhibition of IN catalytic activities, IC₅₀ (μM) GOLD Example Structure 3′-processing Strand transfer Score OT33

>>100 95 ± 11 64

TABLE 42 Inhibition of HIV-1 Integrase Catalytic Activities and GOLD Score of Representative Compounds Exemplified by Formula 38 Inhibition of IN catalytic activities, IC₅₀ (μM) GOLD Example Structure 3′-processing Strand transfer Score OT8

31 ± 16 14 ± 4  41

TABLE 43 Inhibition of HIV-1 Integrase Catalytic Activities and GOLD Score of Representative Compounds Exemplified by Formula 39 Inhibition of IN catalytic activities, IC₅₀ (μM) GOLD Example Structure 3′-processing Strand transfer Score OT32

72 ± 8 57 ± 22 47 SR44

32 16

TABLE 44 Inhibition of HIV-1 Integrase Catalytic Activities and GOLD Score of Representative Compounds Exemplified by Formula 40 Inhibition of IN catalytic activities, IC₅₀ (μM) GOLD Example Structure 3′-processing Strand transfer Score OT2

>100 95 ± 9 36 RT25

>100 100

TABLE 45 Inhibition of HIV-1 Integrase Catalytic Activities of Representative Compounds Exemplified by Formula 41 Inhibition of IN catalytic activities, IC₅₀ (μM) Example Structure 3′-processing Strand transfer RT3

45 69 RT29

30 70 RT31

57 >100 RT30

>100 >100 Pharmacophore Models: Several common feature pharmacophore models were generated using a set of known HIV-1 integrase inhibitors. The top-ranking pharmacophores are expected to identify the hypothetical orientation of the active compounds and the common binding features interacting with the target. All training set inhibitors were somewhat structurally diverse but possessed some common chemical features, and comparable inhibitory potencies. The pharmacophore models were generated using low energy conformation as well as using conformations similar to the crystallographically determined conformation of known integrase inhibitors. On the basis of the structural information from these known inhibitors and the active site of the HIV-1 integrase, a set of features were selected to be present in the pharmacophore generation experiment. The chemical features considered in the pharmacophore model generation run were H-bond donor (HBD), H-bond acceptor (HBA), Hydrophobic aromatic (HRA), Aromatic ring center (AR), and Negatively ionizable (NI) features. The most potent known HIV-1 integrase inhibitor (GS-9137 (5)) is mapped onto the best ranked pharmacophore model. All the pharmacophore models that were generated specific to HIV-1 integrase are given in Table 46 along with feature combinations and ranking scores. Database Search: The highest ranked common feature pharmacophore model 1 was used as search query to retrieve compounds with novel chemical structure and desired chemical features from an in-house multi-conformer Catalyst-formatted database consisting of ˜5,000,000 compounds. Drug-like properties of the retrieved hits from the database search were calculated using Accord for Excel. Docking and Virtual Screening: The subunit B of the core domain X-ray structure of Integrase (PDB 1BIS) in which all the active site amino acid residues were resolved was chosen for our docking and virtual screening purpose. A Mg²⁺ ion was placed in the active site between carboxylate oxygen atoms of amino acid residues D64 and D116 considering the geometry of the Mg²⁺ ion that was present in the subunit A of IN in PDB 1BIS and subunit A in IN-5CITEP complex X-ray structure (PDB 1SQ4). All the water molecules present in protein were removed and proper protonation states were assigned for acidic and basic residues of the protein. Docking was performed using version 1.2 of the GOLD (Genetic Optimization for Ligand Docking) software package. GOLD is an automated ligand docking program that uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the receptor. The algorithm was tested on a dataset of over 300 complexes extracted from the Brookhaven Protein DataBank. GOLD succeeded in more than 70% cases in reproducing the experimental bound conformation of the ligand. GOLD requires a user defined binding site. It searches for a cavity within the defined area and considers all the solvent accessible atoms in the defined area as active site atoms. A 20 A radius active site was defined considering the carboxylate oxygen (OD1) atom of residue D64 as the center of the active site. All the compounds retrieved by the pharmacophore models were docked into the active site of IN. On the basis of the GOLD fitness score, for each molecule a bound conformation with high fitness score was considered as the best bound-conformation. All docking runs were carried out using standard default settings with a population size of 100, a maximum number of 100,000 operations, and a mutation and crossover rate of 95. The fitness function that was implemented in GOLD consisted basically of H-bonding, complex energy and ligand internal energy terms. Materials. Chemicals, and Enzymes: All compounds were dissolved in DMSO and the stock solutions were stored at −20° C. The [³²P]-ATP was purchased from either Amersham Biosciences or ICN. Preparation of Oligonucleotide. Substrates: The oligonucleotides 21 top, 5′-GTGTGGAAAATCTCTAGCAGT-3′ (SEQ ID NO:1) and 21 bot, S′-ACTGCTAGAGATTTTCCACAC-3′ (SEQ ID NO:2) were purchased from Norris Cancer Center Core Facility (University of Southern California) and purified by UV shadowing on polyacrylamide gel. To analyze the extent of 3′-processing and strand transfer using 5′-end labeled substrates, 21 top was 5′-end labeled using T4 polynucleotide kinase (Epicentre, Madison, Wis.) and v [³²P]-ATP (Amersham Biosciences OT ICN). The kinase was heat-inactivated and 21 bot was added in 1.5-molar excess. The mixture was heated at 95° C., allowed to cool slowly to room temperature, and run through a spin 25 mini-column (USA Scientific) to separate annealed double-stranded oligonucleotide from unincorporated material. Integrase Assays: To determine the extent of 3-processing and strand transfer, wild-type IN was preincubated at a final concentration of 200 nM with the inhibitor in reaction buffer (50 mM NaCl, 1 mM HEPES, pH 7.5, 50 μM EDTA, 50 μM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnC12, 0.1 mg/ml bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide, and 25 μM MOPS, pH 7.2) at 30° C. for 30 min. Then, 20 nM of the 5′-end ³²P-labeled linear oligonucleotide substrate was added, and incubation was continued for an additional one hour. Reactions were quenched by the addition of an equal volume (16 μl) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue). An aliquot (5 μl) was electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M tris-borate pH 8.3, 2 mM EDTA, 20% acrylamide, 8M urea). Gels were dried, exposed in a Phosphorlinager cassette, and analyzed using a Typhoon 8610 Variable Mode Imager (Amersham Biosciences) and quantitated using ImageQuant 5.2. Percent inhibition (% 1) was calculated using the following equation: %I=100×[1−(D−C)/(N−C)] where C, N, and D are the fractions of 21-mer substrate converted to 19-mer (3-processing product) or strand transfer products for DNA alone, DNA plus IN, and IN plus drug, respectively. The 1050 values were determined by plotting the logarithm of drug concentration versus percent inhibition to obtain concentration that produced 50% inhibition. Anti-HIV Assays in Cultured Cells: The anti-HIV activity was evaluated in human T cell line CEM-SS infected with HIV-1 as described by Weislow et al. 45 In brief, cells were plated in 96-well plates at 5×10³ cells/well and infected with HIV-1RF (MOI=0.3). Serial dilutions of compounds were then immediately added to the cells in a final volume of 200 μl. In each experiment, AZT and dextran sulfate were included as control compounds for anti-HIV activity. The cells were maintained at 37° C. with 5% C02-containing humidified air for 6 days. Cell viability was quantified by absorbance at 450 nm after 4 h incubation with 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) at 0.2 mg/ml. Antiviral activity was graded based on the degree of anti-HIV protection as active (80-100% protection), moderate (50-79% protection) and inactive (0-49% protection). Toxicity of the compounds was determined simultaneously on the same plate in uninfected CEM-SS cells.

TABLE 46 Pharmacophore models generated using the training set compounds (1-5) (All chemical features found in the training set compounds considered for pharmacophore generation except the H-bond donor feature) Hypothesis Features Combinations ^(a) Ranking Score HT5NoDF.01 HRA1, HRA2, HBA, NI 53.40 HT5NoDF.02 RA1, HRA1, HBA, NI 53.09 HT5NoDF.03 RA1, HRA1, HBA, NI 53.09 HT5NoDF.04 RA1, HRA1, HBA, NI 53.07 HT5NoDF.05 RA1, HRA1, HBA, NI 53.07 HT5NoDF.06 RA1, RA2, HBA, NI 52.71 HT5NoDF.07 RA1, RA2, HBA, NI 52.71 HT5NoDF.08 RA1, RA2, HBA, NI 52.71 HT5NoDF.09 RA1, RA2, HBA, NI 52.71 HT5NoDF.10 HRA1, HRA2, HBA, NI 51.73 ^(a) HRA1-2:—Hydrophobic Aromatic; HBA:—H-Bond Acceptor; RA1-2:—Ring Aromatic; NI—Negatively Ionizable Feature

TABLE 47 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 1 Com- IN inhibition activity pound Structure 3′-processing Strand transfer AV15

9 ± 2   4 ± 0.5 AV15A11

24 ± 16 20 ± 13 AV15A13

AV15A44

>100    68 ± 22 AV15A45

91 ± 16 39 AV15A48

>100    >100    AV15A54

>100    >100    AV15A1

>>100      96 ± 6  AV15A3

? ? AV15A37

>100    >100    AV15A43

63 ± 7  24 ± 11 AV15A27

42 ± 24 26 ± 12 AV15A16

>100    >100    AV15A26

>>100      88 AV15A28

>100    >100    AV15A39

>100    >100    AV15A42

89 ± 19 ? AV15A4

>>100      96 ± 6  SA1

59 ± 1  9 ± 5 AV15A40

90 ± 18 46 ± 23 AV15A41

>100    >100    AV15A36

>100    56 ± 22 AV15A38

>100    49 ± 12 AV15A29

>100    100  AV15A5

>>100      88 ± 21 AV15A20

>>100      95 AV15A47

>100    >100    AV15A35

>>100      86 AV15A6

76 24 AV15A2

>>100      90 ± 17 T36

12 ± 6  6.5 ± 1   X114

40 ± 10 43 ± 6  AV15A22

23 ± 3  15 ± 4  AV15A32

>100    >100    AV15A12

74 ± 32 50 ± 25 AV15A23

89 ± 20 62 ± 7  AV15A25

>100    >100    AV15A7

>>100      17, 100 AV15A9

25 ± 10 14 ± 4  AV15A10

76 30 AV15A31

34 24 AV15A46

>100    >100    AV15A52

100  51 AV15A55

AV15A14

16 ± 8  13 ± 5  AV15A15

12 ± 6  6.5 ± 1   AV30

10 ± 3  4 ± 1 AV15A21

10 ± 5  7 ± 2 AV15A24

5 ± 3 3.4 ± 2   AV15A33

>>100      AV15A18

30 ± 12 17 ± 5  AV15A19

>>100      >>100     

TABLE 48 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 41 GOLD Example Structure Score NAV1

48 NAV2

56 NAV3

45 NAV4

40 NAV5

42 NAV6

41 NAV7

50 NAV8

50 NAV9

45 NAV10

42 NAV11

48 NAV12

48

TABLE 49 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 42 IN inhibition activity GOLD Compound Structure 3′-processing Strand transfer Score M28 (X155)

93 ± 12 67 ± 28 X81

84 ± 23 69 ± 36 T38

25 20 AS16

88 40 ± 5  AS143

60 ± 3  42 ± 10 AS134

47 ± 7  26 ± 9  AS139 BAS 02234865

27 ± 10 8 ± 2 60.30 AS161

57 ± 27 30 ± 7  AS171

45 ± 8  43 ± 26 M28A2

64 ± 28 43 ± 8  M28A6

56 ± 10 39 ± 3  M28A7

>100 78 ± 10 M28A11

>100 90 ± 17 M28A10

44 ± 4  21 ± 4  M28A18

>100 33 ± 25 M28A8

57 ± 10 21 ± 10 M28A21

>100 90 ± 10 M28A22

>100 83 ± 12 AS139noNO2

63.29 AS139SnoNO2

72.21 AS139S1

71.08 AS139S2

69.26 AS139S3

69.86 AS139S3NH2

72.38 AS139S4

69.61 AS139S4NH2

71.09 AS139S5

68.80 AS139S5NH2

67.50 AS139S6

68.18 AS139S6OH

66.03 AS139S3A2

73.18 AS139S3A3

73.96 AS139S3A4

75.27 AS139S3A5

78.52 AS139S3A5A

73.56 AS139S3A1

74.28 AS139S3A6

72.60 AS139S3A7

72.41 AS139S3A8

70 AS139S7

72.78 AS139S7NH2

70.99 AS139S7

70.55 AS139S7ME

69.11

TABLE 50 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 43 IN inhibition activity Strand Compound Structure 3′-processing transfer AS9

5 ± 2 3 ± 1 AS90

21 ± 6  9 ± 7 AS120

23 ± 6  12 ± 8  AS230

9 ± 1 8 ± 4 AS9A1

64 ± 4  58 ± 2  AS9A2

18 ± 1  17 ± 1  AS9A3

51 ± 5  27 ± 8  AS9A4

21 ± 2  17 ± 1  AS9A5

<100 <100 AS9A6

8 ± 1 6 ± 1 AS9A7

49 ± 1  31 ± 2  AS9A8

68 ± 3  61 ± 6  AS9A9

11 ± 3  5 ± 1 AS9A10

22 ± 1  21 ± 1  AS9A11

15 ± 3  9 ± 2 MC41

>>100 >>100 X244

60 ± 11 51 ± 3  GLD38

>100 >100 GLD39

>100 >100 OT10

>100 >100 HD39

>100 >100 MPD6

LX6

>100 >100 LX7

>100 >100

TABLE 51 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 44 IN inhibition activity Strand Pharmacophore Compound Structure 3′-processing transfer Fit Value GLD2

18 ± 4  5 ± 3 2.3 GLD2A2

98 ± 10 60 ± 12 GLD2A4

>100 >100 GLD2A10

>100 >100 GLD2A3

>100 >100 GLD2A18

>100 >100

TABLE 52 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 45 IN inhibition activity Strand Pharmacophore Compound Structure 3′-processing transfer Fit Value GLD44

14 ± 6  5 ± 4 1.7 GLD45

82 ± 13 43 ± 12 1.7

TABLE 53 HIV-1 Integrase Inhibitory Activities of Structurally Diverse Compounds IN inhibition activity Strand Pharmacophore Compound Structure 3′-processing transfer Fit Value GLD19

>100 52 ± 8  1.7 GLD20

>100 46 ± 12 2.9 GLD25

42 ± 10 18 ± 3  3.5 GLD28

48 ± 19 23 ± 4  3.1 GLD31

92 ± 17 66 ± 17 3   GLD32

>100 34 ± 7  3.8 GLD59

63 ± 22 17 ± 2   1.53

TABLE 54 HIV-1 Integrase Inhibitory Activities of Representative Compounds Exemplified by Formula 46 IN inhibition activity Strand Pharmacophore Compound Structure 3′-processing transfer Fit Value GLD12

14 ± 7  5 ± 3 3.1 GLD12A2

5 ± 2 4 ± 2  2.31 GLD12A4

7 ± 6 4 ± 2  2.44 GLD12A3

75 ± 28 59 ± 13  2.31 GLD12A5

11 ± 3  13 ± 11  2.30 GLD12A1

61 ± 8  65 ± 8   2.30 GLD12D1

GOLD Score 50.05 GLD12D2

48.03 GLD12D3

48.40 GLD12D4

50.13 GLD12D5

49.27 GLD12D6

46.59 GLD12D7

49.55 GLD12D8

48.00 GLD12D9

50.64 GLD12D10

42.40 GLD12D11

41.65 GLD12D12

41.37 GLD12D13

47.57 GLD12D14

50.30 GLD12D15

GLD12D16

GLD12D17

TABLE 55 HIV-1 Integrase Inhibitory Activities and Pharmacophore Fit Values of Compounds Designed Based on the GLD12 Scaffold Pharmacophore Compound Structure Fit Value GLD12DA

2.46 GLD12D1F

3.86 GLD12D1FA

3.23 GLD12D1FB

2.50 GLD12D1FD

2.01 GLD12D1FE

3.75 GLD12D1FF

3.69 GLD12D1FG

GLD12D1FH

2.74 GLD12D1FI

3.88 GLD12D1FIA

3.88 GLD12D1FIB

3.88 GLD12DNM1

GLD12DNM2

TABLE 56 Representative Compounds Exemplified by Formula 47 Com- pound Structure 3FS1

3FS2

3FS3

3FS4

3FS5

3FS6

3FS7

3FS8

3FS9

3FS10

TABLE 57 Representative Compounds Exemplified by Formula 48 Compound Structure 3FSN1

3FSN2

3FSN3

3FSN4

3FSN5

3FSN6

3FSN7

REFERENCES

-   Deng et at (2007) Biorganic & Medicinal Chemistry 15:4985-5002. -   Dayam et al. (2008) J. Med. Chem., 51, 1136-1144.

All publications cited herein are incorporated by reference in their entirety. While the foregoing has been described in considerable detail and in terms of preferred embodiments, these are not to be construed as limitations on the disclosure. Modifications and changes that are within the purview of those skilled in the art are intended to fall within the scope of the invention. 

1. A method of inhibiting HIV-1 integrase, comprising contacting a composition comprising

wherein R₁ is selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, alkoxy, carboxyl, carbonyl, nitro, cyano, amino, amide, sulfonyl, sulfonamide, nitro, substituted aliphatic, heteroaliphatic, aryl, and heteroaryl, wherein X is selected from the group consisting of CH or N, or a pharmaceutically acceptable salt thereof with an HIV-1 integrase, thereby inhibiting the activity of the HIV-1 integrase.
 2. The method of claim 1, wherein X is N.
 3. The method of claim 2, wherein R₁ is H.
 4. The method of claim 1, wherein the composition further comprises a carrier.
 5. A method of inhibiting HIV-1 integrase, comprising contacting a composition comprising

or a pharmaceutically acceptable salt thereof, with an HIV-1 integrase, thereby inhibiting the activity of the HIV-1 integrase. 